medicines

Short Note
Anti-Bacterial Activity of Phenolic Compounds
against Streptococcus pyogenes
Sabrina Macé 1 , Lisbeth Truelstrup Hansen 2,† and H.P. Vasantha Rupasinghe 1,3, *
1 Department of Plant, Food, and Environmental Sciences, Faculty of Agriculture, Dalhousie University,
Truro, NS B2N 5E3, Canada; sabmace@gmail.com
2 Department of Process Engineering and Applied Science, Faculty of Engineering, Dalhousie University,
Halifax, NS B3H 4R2, Canada; litr@food.dtu.dk
3 Department of Pathology, Faculty of Medicine, Dalhousie University, Halifax, NS B3H 4H7, Canada
* Correspondence: vrupasinghe@dal.ca; Tel.: +1-902-893-6623
† Current address: National Food Institute, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark.

Academic Editor: Eleni Skaltsa

Received: 1 April 2017; Accepted: 26 April 2017; Published: 1 May 2017

Abstract: Background: Worldwide, Streptococcus pyogenes is the leading cause of bacterial pharyngitis.
To reduce the use of antibiotics, antimicrobial phytochemical-containing remedies, which have
long been in use in traditional medicine, may provide new approaches for management of
streptococcal pharyngitis. The objective of this study was to assess the inhibitory activities of
25 natural phenolic compounds against three strains of S. pyogenes. Methods: After an initial
screening, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration
(MBC) of the nine most effective phenolic compounds were determined. The effect of four
compounds with the lowest MIC and MBC on streptococcal growth and biofilm formation was
also studied. Results: 1,2-Naphthoquinone and 5-hydroxy-1,4-naphthoquinone elicited the greatest
anti-S. pyogenes activities with MICs ranging from 0.39 to 6.25 µg mL−1 and MBCs of 100 µg mL−1 .
Both naphthoquinones inhibited the biofilm formation at concentrations ranging from 12.5 to
50 µg mL−1 . Biofilm reduction and altered bacterial cell structures were visible in scanning electron
microscopy images of naphthoquinone-treated cells. Conclusion: In conclusion, 1,2-naphthoquinone
and 5-hydroxy-1,4-naphthoquinone inhibit S. pyogenes and should be further investigated as
candidates for the management of streptococcal pharyngitis.

Keywords: pharyngitis; strep throat; biofilm; naphthoquinone; infection; disease; polyphenols

1. Introduction
Pharyngitis or bacterial infection of the throat is a common disease, which is often caused by
Streptococcus pyogenes [1,2]. Worldwide, S. pyogenes are responsible for an estimated 600 million cases of
throat infections per year [3]. β-Lactams, such as penicillin, are the preferred antibiotics in the treatment
of S. pyogenes throat infections, with macrolides being used for patients with β-lactam hypersensitivity.
However, while resistance to β-lactams has so far not emerged in S. pyogenes, resistances were found
for macrolides and some quinolones (fluoroquinolone) [3,4]. Moreover, S. pyogenes forms biofilm,
which has been associated with antibiotic treatment failures [5]. Thus, there is an urgent need to
find new potent antibacterial and anti-biofilm agents which could find use in alternative and/or
complementary therapy for streptococcal pharyngitis.
In addition to treatment with prescribed medications, throat lozenges containing chemical
anesthetics and antiseptics are being used by patients in the relief of the discomfort associated with
the infection. The antimicrobial quaternary ammonium compound, dequalinium chloride, is similarly
used in the treatment of common infections of the mouth and throat and incorporated in candy-based

Medicines 2017, 4, 25; doi:10.3390/medicines4020025 www.mdpi.com/journal/medicines

Medicines 2017, 4, 25 2 of 9

lozenge formulations [6]. The antiseptic and local anesthetic hexylresorcinol has also been included as
an active ingredient in throat lozenges [7].
Natural products derived from plants have been used in traditional medicine since the ancient
times and are, now, being widely studied for incorporation into mainstream products. Plant-derived
compounds of interest are mostly secondary metabolites, which possess antimicrobial properties
against microbial pathogens and spoilers [8]. Recently, a diverse range of phytochemical antibacterial
agents has been reported to suppress the growth of S. pyogenes, including polyphenols (i.e., flavonoids)
and terpenes [9–12]. A recent review describes the mechanisms of inhibition of different Streptococcus
species, including S. pyogenes, by phytochemicals through the prevention of the bacterial adherence of
the pharynx, inhibition of glycolytic enzyme and pH drop, reduction of biofilm, and alteration of cell
surface hydrophobicity [2].
The objective of the present study was to identify, among 25 different commercially available
plant-derived phenolic compounds, the ones with anti-S. pyogenes activities, which could be
incorporated in dehydrated honey throat lozenges and similar products.

2. Material and Methods

2.1. Bacterial Strains and Growth Conditions

The experiments used the following human S. pyogenes strains: S. pyogenes ATCC ® 19615 ™,
S. pyogenes ATCC ® 49399 ™ and a clinical isolate obtained from a patient with pharyngitis (Bacteriology,
Queen Elizabeth II Hospital, Halifax, NS, Canada). The strains were routinely cultured in Brain
Heart Infusion (BHI) (Oxoid, Thermo Fisher Scientific, Waltham, MA, USA) media at 37 ◦ C under
aerobic conditions.

2.2. Compounds
Twenty-five phenolic compounds belonging to 12 different classes: three simple phenols
(eugenol, thymol, and pyrocatechol), three isoflavones (daidzin, daidzein, and genistein), one chalcone
(phloretin), one chalcone glycoside (phlorizin), two flavan-3-ols (epicatechin and epigallocatechin
gallate, EGCG), two flavanones (naringenin and hesperidin), two flavones (flavone and naringin),
two flavonols (myricetin and quercetin hydrate), one flavonol glucoside (quercetin-3-O-glucoside),
one hydroxybenzoates (gallic acid), three hydroxycinnamates (tran-ferulic acid, cinnamaldehyde,
and warfarin), two naphthoquinones (1,2-naphthoquinone and 5-hydro-1,4-naphthoquinone),
one stilbene (resveratrol), one tannin (tannic acid), and two chemical compounds (used as
positive controls), one quaternary ammonium compound (dequalinium chloride), and one substituted
phenol (4-hexylresorcinol) were evaluated for their antibacterial properties. The compounds were
purchased from Sigma-Aldrich Company (Saint-Louis, MO, USA). Before each experiment, the stock
solutions were freshly made to a concentration of 10 mg mL−1 in 100% DMSO.

2.3. Preliminary Screening Using the Micro-Dilution Broth Antibacterial Assay

The inhibitory effect of the phenolic compounds (100 µg mL−1 ) was initially assessed on
S. pyogenes ATCC 19615 and ATCC 49399, using the broth micro-dilution assay as described by Clinical
and Laboratory Standards Institute [13]. Briefly, in a 96-well micro-plate, 2 µL of the 100 mg mL−1
stock solution was added to 98 µL of BHI broth in order to reach a final test compound concentration
of 100 µg mL−1 . Finally, for each strain, 100 µL of standardized inoculum was added, resulting in
an initial bacterial concentration of 5 log10 (CFU mL−1 ). Bacterial inocula in BHI broth (2 µL water)
and BHI broth +1% DMSO were also run as a control. The growth of the bacteria was assessed after
incubation for 24 h at 37 ◦ C by reading the A600 nm using a plate reader (Epoch TM , Biotek, Winooski,
VT, USA). Each test was done in triplicate. In order to determine the most active compounds, a decrease
in the A600 nm of 0.1 or more in the presence of the tested compounds in comparison to the control
(bacteria inocula in BHI +1% DMSO) was considered to be indicative of an inhibition of the growth.
Medicines 2017, 4, 25 3 of 9

2.4. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)
MIC broth micro-dilution assay was performed as described in the previous section, except 2-fold
dilutions were used to yield final test compound concentrations ranging from 0.19 to 100 µg mL−1 ,
on the three S. pyogenes strains. The MIC was determined as the first concentration where the decrease
of A600 nm was significant (p < 0.05) in comparison with the control. To determine MBC, 30 µL from
each well, where no growth was detected—i.e., compound concentration ≥MIC—was spread on
BHI agar plates and incubated for 24–48 h at 37 ◦ C. The lowest compound concentration resulting
in no growth on the agar plate represented the MBC. Each sample was tested in triplicate and each
experiment was repeated three times.

2.5. Time-Kill Assay

The effect of concentrations ranging from 1 to 16 times MIC of the four most active compounds
on the growth of S. pyogenes ATCC 19615 was quantified after 0, 2, 4, 6, 8, 10, and 24 h at 37 ◦ C. At each
time point, an aliquot (100 µL) was pipetted, serially diluted, and plated on BHI agar followed by
enumeration and calculation of the mean log10 CFU mL−1 of the duplicate samples. Each time-kill
experiment was repeated in three biologically independent assays (n = 6).

2.6. Inhibition of Biofilm Formation

The ability of S. pyogenes to form biofilm in the presence of 1,2-naphthoquinone, 5-hydroxy-1-
naphthoquinone, dequalinium chloride, or 4-hexylresorcinol (0.19–100 µg mL−1 ) was assessed
by measuring the metabolic activity of sessile biofilm cells using a 3-(4,5-dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide (MTT) dye assay. After an exposure period of three days at 37 ◦ C,
the planktonic bacterial cells were eliminated by carefully removing the BHI medium from the wells.
Adapted MTT assay was performed as described in Vybrant® MTT Cell Proliferation Assay Kit Protocol
(Thermo Fisher Scientific, Waltham, MA, USA). After incubation for 10 min at 37 ◦ C, the A540 nm was
measured and used to calculate the minimum biofilm inhibitory concentration (MBIC), defined as
the first concentration where the decrease in A540 nm was significant (p < 0.05) in comparison with
the control.

2.7. Scanning Electron Microscopy (SEM)

S. pyogenes ATCC 19615 biofilms formed (72 h at 37 ◦ C) in the presence of MBIC concentrations of
1,2 naphthoquinone, 5-hydroxy 1,4 naphthoquinone, dequalinium chloride, 4-hexylresorcinol were
prepared in microplates. The S. pyogenes biofilms were also grown in BHI broth (water) and BHI
broth +1% DMSO as controls. After incubation, a sample fixation method for SEM [14] adapted
for microplate was performed. After discarding the last solution, the plate was air dried for 2 h in
a fume-hood. The bottom of the wells (location of the biofilms) were then removed with a heated blade
and mounted on aluminum mounts using the carbon adhesive and finally sputter-coated with gold
and examined using a Hitachi S-4700 SEM (Tokyo, Japan).

2.8. Statistical Analysis

During the determination of the MIC and MBIC, statistically significant differences (Student t-test
(paired, two tailed), p < 0.05) between test compound concentrations and controls were determined
using the Student t-test on the A600 nm or A540 nm data, respectively.

3. Results
The results of the screening test against two S. pyogenes strains, ATCC 19615 and ATCC 49399
showed that nine compounds exhibited antibacterial activity against at least one strain at the
concentration of 100 µg mL−1 . Six of them, phlorizin, naringenin, myrcetin 1,2-naphthoquinone,
5-hydroxy-1,4-naphthoquinone, and resveratrol inhibited both strains like the two positive controls.
Medicines 2017, 4, 25 4 of 9

Medicines 2017, 4, 25 4 of 8
Flavone and thymol affected the growth of strain ATCC 19615 while cinnamaldehyde inhibited only
ATCC 49399.
Flavone and thymol affected the growth of strain ATCC 19615 while cinnamaldehyde inhibited only
The MIC of those nine phytochemicals (Table 1) varied from 0.39 µg mL−1 to >100 µg mL−1 and the
ATCC 49399.
MBC from 100 µg mL−1 to >100 µg mL−1 with some sensitivity/resistance variations among strains.
The MIC of those nine phytochemicals (Table 1) varied from 0.39 µg mL−1 to >100 µg mL−1 and
1,2-Naphthoquinone and−15-hydroxy-1,4-naphthoquinone exhibited the best inhibitory effect with
the MBC from 100 µg mL to >100 µg mL−1 with some sensitivity/resistance variations among strains.
similar MIC values relative to the positive controls and were selected for further analysis of their effect
1,2-Naphthoquinone and 5-hydroxy-1,4-naphthoquinone exhibited the best inhibitory effect with
on bacterial growth and biofilm formation.
similar MIC values relative to the positive controls and were selected for further analysis of their
The four compounds that higher concentrations increased the lag phase and the bacterial
effect on bacterial growth and biofilm formation.
log10 -reduction (Figure 1). The presence of low concentrations (<12.5 µg mL−1 ) of 1,2-naphthoquinone
The four compounds that higher concentrations increased the lag phase and the bacterial log10-
delayed growth during the first 10 h but after 24 h the bacterial concentration reached around
reduction (Figure − 1
1). The presence of low concentrations (<12.5
− 1
µg mL−1) of 1,2-naphthoquinone
8 log10 CFU mL (Figure 1A). A concentration of 25 µg mL displayed a bacteriostatic effect
delayed growth during the first 10 h but after 24 h the bacterial concentration reached around 8 log10
for the first 10 h, after which growth resumed to reach 5 log10 CFU mL−1 after 24 h. At 50 µg mL−1 ,
CFU mL−1 (Figure 1A). A concentration of 25 µg mL−1 displayed a bacteriostatic effect for the first 10
the growth was inhibited and a bacterial log10 -reduction −1of more than 3.5 log10 CFU mL−1 observed
h, after which growth resumed to reach 5 log10 CFU mL after 24 h. At 50 µg mL , the growth was
−1
after 24 h.
inhibited and a bacterial log10-reduction of more than 3.5 log10 CFU mL−1 observed after 24 h.

Figure1.1.The
Figure Theeffect
effectofof several
several concentrations
concentrations of four
of the the four compounds
compounds on theongrowth
the growth of S. pyogenes
of S. pyogenes ATCC
ATCC ◦
19615 at 37 °C: (A) 1,2-naphthoquinone (MIC: − 13.125 µg mL
19615 at 37 C: (A) 1,2-naphthoquinone (MIC: 3.125 µg mL ); (B) 5-hydroxy-1,4-naphthoquinone−1); (B) 5-hydroxy-1,4-

naphthoquinone
(MIC: −1 ); (C)
1.56 µg mL(MIC: 1.56 µg mL−1); (C)
dequalinium dequalinium
chloride chloride
(MIC: 0.39 µg mL −1 ); and
(MIC: 0.39 (D)
µg mL −1); and (D) 4-
4-hexylresorcinol
hexylresorcinol
(MIC: 1.56 µg mL − 1
(MIC: 1.56 µg mL
). Arrows −1 ). Arrows
indicate indicate
that plate countthat platewere
values countbelow
valuesthe
were below the
detection detection
threshold of
threshold
1.22 of 1.22
log10 CFU −110
mLlog . CFU mL−1.

Administrationofof
Administration 1.56
1.56 µgµg
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−1 5-hydroxy-1,4-naphthoquinone caused populations to remain under
6under
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than 7than
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was was reached (Figure
−1 −1
−1 for h; however, after 24 h more reached (Figure 1B).
1B). A bacteriostatic effect was similarly observed with 3.125 µg mL − −1 5-hydroxy-1,4-naphthoquinone
1
A bacteriostatic effect was similarly observed with 3.125 µg mL 5-hydroxy-1,4-naphthoquinone
where bacterial
where bacterial counts
counts were
were under
under 66 log
log1010CFU
CFUmLmL−after
−1 24 24
1 after h. Using
h. Using concentrations of 6.25
concentrations and and
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− 1 stayed around 4–5 log 10 CFU mL−1 for 10 h − 1and
12.5 µg mL , numbers stayed around 4–5 log10 CFU mL for 10 h and then dropped about then dropped about 2 log 10 CFU

2mL
−1. With 25 µg mL−1, the bacterial concentrations underwent a 2 log10-reduction after 10 h and
log10 CFU mL−1 . With 25 µg mL−1 , the bacterial concentrations underwent a 2 log10 -reduction after
10 h and the
reached limitthe
reached of detection (>1.22 log
limit of detection 10 CFU mL−1) after 24
(>1.22 log10 CFU mL−1h. ) after 24 h.
For the dequalinium chloride (Figure
For the dequalinium chloride (Figure 1C), the 1C), the same
same trend
trend ofof inhibition
inhibition was
was observed
observed where
where the
the
lowest test concentrations of 0.39 µg mL −1 permitted growth and a twice-higher concentration of 0.78
lowest test concentrations of 0.39 µg mL−1 permitted growth and a twice-higher concentration of
µg mL−1 displayed a bacteriostatic effect. After 24 h, the concentrations of 1.56 and 3.125 µg mL−1
0.78 µg mL−1 displayed a bacteriostatic effect. After 24 h, the concentrations of 1.56 and 3.125 µg mL−1
induced more than 2.5 log10 CFU mL−1 and 6.25 µg mL−1 caused a >4 log10-reduction (below the
detection limit).
Medicines 2017, 4, 25 5 of 9

induced more than 2.5 log10 CFU mL−1 and 6.25 µg mL−1 caused a >4 log10 -reduction (below the
detection limit).
The concentrations of 4-hexylresorcinol ranging from 1.56 to 6.25 µg mL−1 caused a growth
delay and a small log10 -reduction (under 1 log10 CFU mL−1 ) after 24 h. However, the concentration of
mL−1 induced
25 µg Medicines a log10 -reduction of more than 3 log10 CFU mL−1 after 24 h (Figure 1D).
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for 4-hexylresorcinol µg mL
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for
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1,2-naphthoquinone,
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−1 for 4-hexylresorcinol
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Dequalinium
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Dequalinium chloride displayed the 1).
displayed
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and
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from
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andlowestfrom MBICMBIC
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to
ranging
ranging25 µg from
from
25 µgfrom mL
mL−1 0.39–0.78
0.39–0.78 µg
µg mL
4-hexylresorcinol
mL
−1 for 4-hexylresorcinol
−1
0.39–0.78
−1 . The
The
µg mL−1. The sensitivity
−1 sensitivity
(Table
sensitivity
(Table 1). to
1).
to
to
5-hydroxy-1,4-naphthoquinone,
Dequalinium chloride
5-hydroxy-1,4-naphthoquinone,
Dequalinium displayed and
the
and from
lowest
from 12.5
MBIC to
12.5 the
to 25
25 µg
ranging µg mL
from for
for 4-hexylresorcinol
0.39–0.78
mLdepended µg mL ... The
4-hexylresorcinol (Table
The sensitivity
sensitivity
(Table 1).
to
1).
the inhibitionchloride
the inhibition of
of biofilmdisplayed
formation the lowest
varied MBIC
among
MBICtheranging
strainsfrom
and 0.39–0.78
0.39–0.78 µg onmL
on the to
−1compounds. ATCC
Dequalinium
the inhibition
Dequalinium
the inhibition of biofilm
chloride
biofilm
chloride
of formation
displayed
formation
displayed
biofilm formation thevaried
the
varied among
lowest
among
lowest
varied MBIC
among the strains
ranging
strains
ranging
the strains and
from
and
from
and depended
depended
0.39–0.78
depended µg
on
µg
on the−1−1
mL
the
mL
the
−1compounds.
compounds.
. The
The
compounds. ATCC
The sensitivity
sensitivity
ATCC
ATCC to
to
Dequalinium
the inhibition
Dequalinium
the inhibition chloride
of biofilm
chloride
of biofilmdisplayed
formation
displayed
formation the
the lowest
varied
lowest
varied MBIC
among
MBIC ranging
the
ranging from
strains and
from 0.39–0.78
depended
0.39–0.78 µg
on
µg mL
the
mL −1 . sensitivity
compounds.
. The ATCC
sensitivity to
to
19615
19615
the
19615
Table
the
19615
was
was
1.
was
more
more
inhibition
was more
Minimum
inhibition
more of sensitive
of sensitive
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sensitive
sensitive
to
to
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biofilm varied among
to 1,2-naphthoquinone
1,2-naphthoquinone
formation
1,2-naphthoquinone
formation
to concentration
varied among
1,2-naphthoquinone
the
the strains
but
but
among (MIC,
but
the
but
more
more
strains
moreµg
and
and−depended
resistant
resistant
strains
more mL
and 1 ),to
resistant to
depended
to
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resistant to
on
on the compounds.
4-hexylresorcinol
4-hexylresorcinol
thebactericidal
compounds.
4-hexylresorcinol
minimumon the compounds.
4-hexylresorcinol
than
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ATCC
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two
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19615
the inhibition
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biofilm formation varied
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to 1,2-naphthoquinone
1,2-naphthoquinone the
the strains
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and depended
more resistant
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on the
the compounds.
to 4-hexylresorcinol
4-hexylresorcinol ATCC
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19615
other
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was
strains
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wasmL
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(Table
(Table
more
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more
strains 1) formation
−1 )sensitive
1)
sensitive
1)
sensitive
(Table 1)
to
to
among but
to 1,2-naphthoquinone
1,2-naphthoquinone
strains
more
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but
depended
more resistant
more resistant to compounds.
to 4-hexylresorcinol
to 4-hexylresorcinol than ATCC
the two
than the
than twomL−1 ) of
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(MBC,
19615
other
19615
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µg
was more
strains
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more
(Table
of1)
11 compounds
sensitive
1)
sensitive to
to and minimum
1,2-naphthoquinone
1,2-naphthoquinone but
but more
more inhibitory
resistant
resistant to
to concentration than
4-hexylresorcinol
4-hexylresorcinol (MBIC,
than theµg
the two
two
other
other strains
strains (Table
(Table 1)
1)compounds against three different−1−1strains of S. pyogenes.
the four
other
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Table 1. active
1.(Table
strains
Table (Table
Minimum
Minimum 1)
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(MIC, µgµg mL
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), minimum
minimum bactericidal
bactericidal concentration
concentration
Table 1.
Table 1. Minimum
Minimum inhibitory
inhibitory concentration
concentration (MIC,
(MIC, µgµg mL
mL−1 ), minimum
−1), minimum bactericidal
bactericidal concentration
concentration
Table
(MBC, 1.
µg Minimum
mL −1)) of
inhibitory concentration (MIC, µg mL
mL ), minimum bactericidal concentration
of 11 compounds and
and minimum biofilm inhibitory concentration (MBIC, µg
µg mL −1)) of
−1 −1
Table
(MBC, 1.
Table
(MBC, µgMinimum
1.
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Minimum
mL −1 ) of
of 11inhibitory
11 compounds
inhibitory
compounds concentration
and minimum
concentration
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(MIC, µg
biofilm
µg mL
biofilm mL
−1),
−1), minimum
inhibitory
inhibitory minimum bactericidal
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(MBIC, mL−1
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µg mL −1 of
) of
of
Table
(MBC,
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Table
Table 1.
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Minimum
Minimum )
) of 11
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and minimum
and minimum
minimum
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concentration (MIC, µg
biofilm
biofilm
(MIC,
(MIC, µg
µg
−1),
inhibitory
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mL
mL −1
−1 minimum
),), of bactericidal
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(MBIC,
bactericidal
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µg
µg mL
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−1)) of
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Streptococcus
the
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fourµgmost
mL
most active
)
active compounds
compounds
compounds against
and
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threeATCCbiofilm
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inhibitory
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19615 S.
of S. pyogenes.
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ATCC (MBIC,
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))) of
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the four most −1
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the four
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Streptococcus
four
Streptococcus
Compounds most active
most active compounds
active compounds against
compounds against
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againstMIC
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different
different
different
MBC
strains
strains
strains
MBIC
of
of
of
S.
S.
S.
pyogenes.
pyogenes.
pyogenes.
MIC MBC MBIC MIC
Streptococcus
the four
four most
theStrains
Streptococcus
Pyogenes most active
active compounds
compounds
Chemical against
against three
threeATCC 19615
different
19615 strains
different
ATCC
ATCC 19615 strains of
of S. ATCC 49399
S. pyogenes.
pyogenes.
ATCC 49399
ATCC 49399 A Clinical
A Clinical
A Clinical IsolateMBC
Isolate
Isolate
MBIC
Streptococcus
Pyogenes Strains
Streptococcus
Pyogenes Strains Chemical Structure
Structure
Chemical Structure
Structure ATCC 19615
ATCC 19615
19615 ATCC 49399ATCC 49399
49399A Clinical Isolate
A Clinical
Clinical Isolate
Isolate
Pyogenes Strains
Streptococcus
Compounds Chemical MIC ATCC
MBC ATCC A
Pyogenes
Pyogenes Strains
Streptococcus
Compounds
Strains
Streptococcus
Compounds
Chemical
Chemical Structure
Structure MIC
MIC ATCC
MBC
ATCC 19615 MBIC
MBC19615 MBIC
MBIC
MIC
MIC
MIC
MBC
ATCC 49399MBIC
MBC49399
MBC
ATCC MBIC
MBIC
MIC
MIC
MIC A
MBC
A Clinical
Clinical
MBC Isolate
MBC
MBIC
Isolate
MBIC
MBIC
Streptococcus
Pyogenes Strains
Compounds Chemical Structure
Structure MIC MBC
ATCC 19615 MBIC MIC MBC
ATCC 49399 MBIC MICA MBC MBIC
Compounds
Pyogenes Strains Chemical MIC ATCC
MBC 19615 MBIC MIC ATCC
MBC 49399MBIC MIC A Clinical
Clinical
MBC Isolate
Isolate
MBIC
Compounds
Pyogenes
Pyogenes Strains
Strains Chemical Structure
Chemical Structure MIC MBC MBIC MIC MBC MBIC MIC MBC MBIC
Compounds MIC MBC MBIC MIC MBC MBIC MIC MBC MBIC
Compounds
Phlorizin
Compounds
Compounds
MIC
100
MIC
MIC >100 a MBIC
MBC
MBC
MBC
MBIC
MBICnd
MIC
MIC
MIC
MBC
100
MBC >100
MBC MBIC
MBIC
MBIC
MIC
nd MBC
MIC
MIC
MBC
MBC100 MBICMBIC
MBIC>100 nd
Phlorizin
Phlorizin 100
100 >100 a
>100 aaa nd
nd 100
100 >100
>100 nd
nd 100
100 >100
>100 nd
nd
Phlorizin
Phlorizin 100
100 >100
>100 nd
nd 100
100 >100
>100 nd
nd 100
100 >100
>100 nd
nd
Phlorizin
Phlorizin 100
100 >100 aa
>100 nd
nd 100
100 >100
>100 nd
nd 100
100 >100
>100 nd
nd
Phlorizin
Phlorizin 100
100 >100
>100 a
a nd
nd 100
100 >100
>100 nd
nd 100
100 >100
>100 nd
nd
Phlorizin
Phlorizin 100
100 >100
>100 aaa nd
nd 100
100 >100
>100 nd
nd 100
100 >100
>100 nd
nd

Naringenin
Naringenin 50
50 >100
>100 nd
nd 50
50>100 >100
nd
nd
50 >100
50 nd
>100 nd
Naringenin
Naringenin 50
50 >100
>100 nd
nd 50
50 >100
>100 nd
nd 50
50 >100
>100 nd
nd
Naringenin
Naringenin 50
50 >100
>100 nd
nd 50
50 >100
>100 nd
nd 50
50 >100
>100 nd
nd
Naringenin 50 >100 nd 50 >100 nd 50 >100 nd
Naringenin
Naringenin 50
50 >100
>100 nd
nd 50
50 >100
>100 nd
nd 50
50 >100
>100 nd
nd
Naringenin
Naringenin 50
50 >100
>100 nd
nd 50
50 >100
>100 nd
nd 50
50 >100
>100 nd
nd

Flavone
Flavone
Flavone 50
50
50 >100
>100
>100 nd
nd
nd >100
>100
>100
>100
>100
>100
nd
nd
nd
25
25 >100
>100
25 >100
nd
nd
nd
Flavone
Flavone 50
50 >100
>100 nd
nd >100
>100 >100
>100 nd
nd 25
25 >100
>100 nd
nd
Flavone
Flavone 50
50 >100
>100 nd
nd >100
>100 >100
>100 nd
nd 25
25 >100
>100 nd
nd
Flavone
Flavone 50
50 >100
>100 nd
nd >100
>100 >100
>100 nd
nd 25
25 >100
>100 nd
nd
Flavone
Flavone 50
50 >100
>100 nd
nd >100
>100 >100
>100 nd
nd 25
25 >100
>100 nd
nd

Myricetin
Myricetin
Myricetin 50
5050 >100
>100
>100 nd
nd nd 100
100 >100
100
>100 nd
>100
nd 100
nd
100 >100
100
>100 nd
>100
nd nd
Myricetin
Myricetin 50
50 >100
>100 nd
nd 100
100 >100
>100 nd
nd 100
100 >100
>100 nd
nd
Myricetin
Myricetin 50
50 >100
>100 nd
nd 100
100 >100
>100 nd
nd 100
100 >100
>100 nd
nd
Myricetin
Myricetin 50
50 >100
>100 nd
nd 100
100 >100
>100 nd
nd 100
100 >100
>100 nd
nd
Myricetin
Myricetin 50
50 >100
>100 nd
nd 100
100 >100
>100 nd
nd 100
100 >100
>100 nd
nd

Thymol
Thymol 25
2525 >100
>100 nd
nd >100
>100 >100
>100 nd
nd ≥100
≥100 >100
>100 nd
nd
Thymol
Thymol
Thymol
Thymol
25
25
25
>100
>100
>100
>100
nd
nd
nd
nd >100
>100
>100
>100
>100
>100
>100
>100
nd
nd
nd
nd
≥100
≥100
≥100
≥100
>100
>100
>100
nd
nd
nd
>100 nd
Thymol 25 >100 nd >100 >100 nd ≥100 >100 nd
Thymol
Thymol 25
25 >100
>100 nd
nd >100
>100 >100
>100 nd
nd ≥100
≥100 >100
>100 nd
nd
Thymol
Thymol 25
25 >100
>100 nd
nd >100
>100 >100
>100 nd
nd ≥100
≥100 >100
>100 nd
nd
1,2-
1,2-
1,2-
1,2- 3.125
3.125 100
100 25
25 6.25
6.25 100
100 50
50 6.25
6.25 100
100 50
50
Naphthoquinone
1,2-
Naphthoquinone 3.125 100 25 6.25 100 50 6.25 100 50
1,2-Naphthoquinone
1,2-
Naphthoquinone
Naphthoquinone
1,2-
3.125
3.125
3.125 100
100100 25
25 25 6.25
6.25 6.25100
100 100
50
50 50
6.25
6.25 6.25
100
100 50
50 100 50
Naphthoquinone
1,2- 3.125 100 25 6.25 100 50 6.25 100 50
Naphthoquinone
1,2-
1,2- 3.125
3.125 100
100 25
25 6.25
6.25 100
100 50
50 6.25
6.25 100
100 50
50
Naphthoquinone
Naphthoquinone 3.125
3.125 100
100 25
25 6.25
6.25 100
100 50
50 6.25
6.25 100
100 50
50
Naphthoquinone
Naphthoquinone
5-Hydroxy-1,4-
5-Hydroxy-1,4-
5-Hydroxy-1,4-
5-Hydroxy-1,4- 1.56
1.56 100
100 12.5
12.5 1.56
1.56 >100
>100 50
50 0.39
0.39 100
100 12.5
12.5
5-Hydroxy-1,
naphthoquinone
5-Hydroxy-1,4-
naphthoquinone 1.56
1.56 100
100 12.5
12.5 1.56
1.56 >100
>100 5050 0.39
0.39 100
100 12.5
12.5
5-Hydroxy-1,4-
naphthoquinone 1.56
1.56 100100 12.5 1.56
12.5 1.56 >100
1.56 50
>100 0.39
50 100
0.39 12.5100 12.5
naphthoquinone
5-Hydroxy-1,4-
naphthoquinone
5-Hydroxy-1,4- 1.56 100 12.5 >100 50 0.39 100 12.5
4-naphthoquinone
naphthoquinone
5-Hydroxy-1,4-
5-Hydroxy-1,4- 1.56
1.56 100
100 12.5
12.5 1.56
1.56 >100
>100 50
50 0.39
0.39 100
100 12.5
12.5
naphthoquinone
naphthoquinone 1.56
1.56 100
100 12.5
12.5 1.56
1.56 >100
>100 50
50 0.39
0.39 100
100 12.5
12.5
naphthoquinone
naphthoquinone

Resveratrol
Resveratrol 100
100 >100
>100 nd
nd 50
50 >100
>100 nd
nd >100
>100 >100
>100 nd
nd
Resveratrol
Resveratrol 100
100 >100
>100 nd
nd 50
50 >100
>100 nd
nd >100
>100 >100
>100 nd
nd
Resveratrol
Resveratrol 100
100 >100
>100 nd
nd 50
50 >100 nd >100 >100 nd
Resveratrol
Resveratrol 100
100 >100
>100 nd nd 50 50>100
>100
nd
>100
nd
>100
nd
>100
>100
>100
>100
nd
>100
nd nd
Resveratrol
Resveratrol 100
100 >100
>100 nd
nd 50
50 >100
>100 nd
nd >100
>100 >100
>100 nd
nd
Resveratrol 100 >100 nd 50 >100 nd >100 >100 nd

Cinnamaldehyde
Cinnamaldehyde >100
>100 >100
>100 nd
nd 50
50 >100
>100 nd
nd 25
25 >100
>100 nd
nd
Cinnamaldehyde
Cinnamaldehyde >100
>100 >100
>100 nd
nd 50
50 >100
>100 nd
nd 25
25 >100
>100 nd
nd
Cinnamaldehyde
Cinnamaldehyde >100
>100 >100
>100 nd
nd 50
50 >100
>100 nd
nd 25
25 >100
>100 nd
nd
Cinnamaldehyde
Cinnamaldehyde
Cinnamaldehyde >100 >100
>100
>100 >100 nd
>100 nd nd 50 50 50>100
>100 >100 nd
nd nd
25
25 >10025
>100 nd
nd>100 nd
Cinnamaldehyde
Cinnamaldehyde >100
>100 >100
>100 nd
nd 50
50 >100
>100 nd
nd 25
25 >100
>100 nd
nd
Dequalinium
Dequalinium
Dequalinium
Dequalinium 0.39
0.39 12.5
12.5 0.78
0.78 0.78
0.78 25
25 0.78
0.78 0.78
0.78 12.5
12.5 0.39
0.39
chloride
Dequalinium
chloride 0.39
0.39 12.5
12.5 0.78
0.78 0.78
0.78 25
25 0.78
0.78 0.78
0.78 12.5
12.5 0.39
0.39
Dequalinium
chloride 0.39 12.5 0.78 0.78 25 0.78 0.78 12.5 0.39
chloride
Dequalinium 0.39 12.5 0.78 0.78 25 0.78 0.78 12.5 0.39
Dequalinium
chloride
Dequalinium
chloride
Dequalinium
Dequalinium 0.39
0.39 12.5
12.5 0.78
0.78 0.78
0.78 25
25 0.78
0.78 0.78
0.78 12.5
12.5 0.39
0.39
chloride
chloride 0.39 12.5
0.39
0.39 12.5 0.78
12.5 0.78 0.78 0.78
0.78 0.78 25
25 25
0.78
0.78 0.78
0.78
0.78 12.50.78 0.39
12.5 12.5
0.39 0.39
chloride
chloride
chloride
4-Hexylresorcinol 1.56 50 25 6.25 50 12.5 12.5 50 12.5
4-Hexylresorcinol
4-Hexylresorcinol 1.56
1.56 50
50 25
25 6.25
6.25 50
50 12.5
12.5 12.5
12.5 50
50 12.5
12.5
4-Hexylresorcinol
4-Hexylresorcinol 1.56
1.56 50
50 25
25 6.25
6.25 50
50 12.5
12.5 12.5
12.5 50
50 12.5
12.5
4-Hexylresorcinol 1.56 50 25 6.25 50 12.5 12.5 50 12.5
4-Hexylresorcinol 1.56
aa MBC was higher than the maximum concentration 50 25 6.25 50 12.5 12.5 50 12.5
4-Hexylresorcinol
a MBC was was higher
4-Hexylresorcinol higher than
than the
the maximum 1.56
maximum concentration
concentration
1.56 50
50 of
of 100
100 µg
25
µg
25 mL
mL
−1 used
6.25
used
−16.25 in this
50
in50this study;
12.5
study;
12.5 nd: not-determined.
12.5
nd:12.5 50
not-determined.
50 12.5
5012.5 12.5
a MBC 50 of
50100 µg
25 mL used in50this study;
12.5 nd: not-determined.
4-Hexylresorcinol
4-Hexylresorcinol 1.56
1.56 −1
25 −16.25 6.25 50 12.5
12.5 12.550
12.5
MBC was
MBC
a was higher
higher than
than the
the maximum
maximum concentration
concentration of
of 100
100 µg
µg mL
mL−1 used in
−1 used in this
this study;
study; nd:
nd: not-determined.
not-determined.
aMBC
MBC
a was
was higher than
than the
highershowed maximum
the the
maximum concentration
concentration of
of 100
100 µg
µg mL −1 used
used in
mL−1ATCC in this
this study;
study; nd:
nd:innot-determined.
not-determined.
aSEM
MBC
SEM
SEM
MBC images
was higher
images
images
was higher than
showed
showed
than the the
the the visual
maximum
the reduction
visualconcentration
visual
maximum of
concentration
reduction of
reduction S.
of
ofofS.
of pyogenes
100
S.100
S. µg mL
pyogenes
pyogenes
µg
−1 used
ATCC
ATCC in19615
this
19615
19615 biofilm
study; nd:
biofilm
biofilm in
in the presence
not-determined.
the presence
the of
presence of
presence of
of
aa
MBC was higher than
thanthe maximum concentration µg mL used
−1 in this study; nd: not-determined.
of of
S.100 mL used in thisin
study; nd:in
innot-determined.
−1
SEM
a MBC
SEM images
was
images showed
highershowed thethe visual
maximum
visual reduction
concentration
reduction pyogenes
of 100
pyogenes ATCC
mL
µgATCC 19615
used
19615 biofilm
this study;
biofilm the
thend:presence
not-determined.
of
the
the SEM
SEM images
the MBIC
MBIC
MBIC of
of the
images
of the showed
the four
four
four the
the visual
compounds
compounds
showed byreduction
by
visual
compounds by strongly of
strongly
reduction
strongly S.
S. pyogenes
reducing
reducing
of the
pyogenes
reducing the ATCC
ATCC 19615
19615 biofilm
the concentration
concentration
concentration of
of the
biofilm
of the in
in the
the presence
the attached
attached
attached cells
presence
cells of
cells and
and
of
and
the
the SEM
MBIC
SEM
MBIC
SEM images
of
of the
images
the
images showed
four
four the visual
compounds
showed the
compounds
showed the by
visual
by
visual reduction
strongly
reduction
strongly
reduction of
of S.
S. pyogenes
reducing
of S. the
pyogenes
reducing the
pyogenes ATCC
ATCC
ATCC 19615
concentration
19615
concentration
19615 biofilm
of the
biofilm
of the
biofilm in
in the
attached
the
attached
in the presence
cells
presence
cells
presence of
and
of
and
of
the
the MBIC
MBIC of
of the
the four
four compounds
compounds by
by strongly
strongly reducing
reducing the
the concentration
concentration of
of the
the attached
attached cells
cells and
and
the
the MBIC
the MBIC of
MBIC of the
of the four
the four compounds
four compounds
compounds byby strongly
by strongly reducing
strongly reducing the
reducing the concentration
the concentration of
concentration of the
of the attached
the attached cells
attached cells and
cells and
and
 Medicines 2017, 4, 25 6 of 9

Medicines 2017, 4, 25 6 of 8
SEM images showed the visual reduction of S. pyogenes ATCC 19615 biofilm in the presence of
the MBIC of the four compounds by strongly reducing the concentration of the attached cells and
modifying their shape (Figure 2). The presence of 1% DMSO in BHI (Figure 2B) reduced the chain
modifying their shape (Figure 2). The presence of 1% DMSO in BHI (Figure 2B) reduced the chain
length of the cocci and biofilm density, and also appeared to shrink the cells slightly in comparison
length of the cocci and biofilm density, and also appeared to shrink the cells slightly in comparison to
to BHI only treatment (Figure 2A). Figure 2C shows that 25 µg mL −1 of 1,2-naphthoquinone reduced
BHI only treatment (Figure 2A). Figure 2C shows that 25 µg mL−1 of 1,2-naphthoquinone reduced
strongly the concentration of the attached cells, leaving only a few long and thin chains comprised of
strongly the concentration of the attached cells, leaving only a few long and thin chains comprised of
shrunken,
shrunken,irregular
irregular cells. Inthe
cells. In thepresence
presenceof of
12.512.5 µg −mL
µg mL of 5-hydroxy-1,4-naphthoquinone
1 of−15-hydroxy-1,4-naphthoquinone (Figure(Figure
2D),
2D), smaller cells aggregated in clusters of chains. Figure 2E presents the effect of
smaller cells aggregated in clusters of chains. Figure 2E presents the effect of dequalinium chloride dequalinium
chloride at 0.78
at 0.78 µg mL−1µg mL−1,induced
, which which induced the formation
the formation of very
of very small small
groups groupscells
of bacterial of bacterial cells and
and completely
completely altered
altered their shape.their shape.ofThe
The MBIC MBIC of 4-hexylresorcinol
4-hexylresorcinol (Figure
(Figure 2F) affected 2F) affected
the chain formationthe chain
of the
formation of the bacteria and only a few small chains containing
bacteria and only a few small chains containing 2–4 cells were observed. 2–4 cells were observed.

Figure
Figure 2. 2.
Scanning
Scanningelectron
electronmicroscopy
microscopy images
images of of the
the effect
effectof
ofMBIC
MBICconcentrations
concentrations ofof the
the compounds
compounds
ononthe
thebiofilm
biofilmformation
formation by strainS.S.pyogenes
by strain pyogenesATCC ATCC 19615
19615 at ◦37
at 37 C: °C: S. pyogenes
S. pyogenes in BHI in (A)
BHIand(A)with
and1%with
1%DMSO
DMSO (B); S. pyogenes with 25 µg −
mL 1 −1 of 1,2 naphthoquinone (C); 12.5 µg mL
(B); S. pyogenes with 25 µg mL of 1,2 naphthoquinone (C); 12.5 µg mL of 5-hydroxy-1,4 − 1 −1 of 5-hydroxy-1,4

naphthoquinone
naphthoquinone(D); (D); 0.78 µgµgmLmL−−11 of
ofdequalinium
dequalinium chloride
chloride (E) (E)
andand
25 µg µg−1mL
25mL of −1 of 4-hexylresorcinol
4-hexylresorcinol (F).
Magnification
(F). Magnification value: I, ×I,1000;
value: II, ×
×1000; II,5000;
×5000; ×1000;
III,III, ×1000; ×15,000;
IV,IV, ×15,000; ×25,000.
V, V, ×25,000.

4. 4.
Discussion
Discussion
Accordingtotothe
According theliterature,
literature, the
the MICs of
of phenolic
phenoliccompounds
compoundsagainst
againstpharyngitis
pharyngitispathogens
pathogens
range from 1.52 to 100 µg mL −1 [9–12]. Among all the phenolic compounds tested in this study,
range from 1.52 to 100 µg mL [9–12]. Among all the phenolic compounds tested in this study, nine
−1

nine compounds
compounds showed showed an anti-Streptococcal
an anti-Streptococcal activity
activity at a concentration
at a concentration lowerlower than
than 100 µg100
mLµg mL−1 .
−1. Gyawali

and Ibrahim. (2014) [8] stated that the hydroxyl (-OH) group in phenolic compounds may cause
bacterial inhibition and described the importance of double bonds (number and position) in relation
to antimicrobial effectiveness. Inferences could be made to the present study as, among the nine
active compounds, the most anti-S. pyogenes naphthoquinones possess two carbonyl groups in an
aromatic ring as part of their quinone structure. This hypothesis is supported by a recent review
Medicines 2017, 4, 25 7 of 9

Gyawali and Ibrahim. (2014) [8] stated that the hydroxyl (-OH) group in phenolic compounds may
cause bacterial inhibition and described the importance of double bonds (number and position) in
relation to antimicrobial effectiveness. Inferences could be made to the present study as, among
the nine active compounds, the most anti-S. pyogenes naphthoquinones possess two carbonyl
groups in an aromatic ring as part of their quinone structure. This hypothesis is supported
by a recent review which associated the oxydo-reduction activity of the quinone structure in
2-hydroxy-1,4-naphthoquinone with the generation of reactive oxygen species (ROS) and damage
of macromolecules such as DNA, proteins, and lipids [15]. Ndi et al. (2007) [11] isolated another
naphthoquinone from Eremophila serrula, which also exhibited antagonism against S. pyogenes strain
ATCC 10389 with low MIC (7.8 µg mL−1 ) and MBC (15.6 µg mL−1 ).
Clinically, most antibacterials are described as potentially being both bactericidal and
bacteriostatic. This was also observed in this study as the tested compounds. Even if a bactericidal
action is preferred in the context of treatment, achieving a bacteriostatic effect may advantageously
inhibit exotoxin production in Staphylococci and Streptococci thereby avoiding induction of the toxic
shock syndrome [16]. In comparison with the kinetics of the tested compounds, rhodomyrtone—the
potent bioactive compound from Rhodomyrtus tomentosa—appears to be slightly more effective:
it exerted a faster bactericidal activity on S. pyogenes (within 5 and 6 h) at lower concentrations
(6.5–12.5 µg mL−1 ) [17]. However, rhodomyrtone (Sigma-Aldrich Co., Saint-Louis, MO, USA) is about
2000–4500 times more expensive than the naphthoquinones tested.
To the best of our knowledge, the anti-biofilm potential of naphthoquinones against S. pyogenes
has not been explored. The inhibition of biofilm formation is an interesting way to prevent the
formation of well-organized attached bacterial biofilms and, thus, pharyngitis. We observed that
the naphthoquinones elicited significant anti-biofilm activity against S. pyogenes, with metabolic
MBICs in the same range as 4-hexylresorcinol and this was confirmed by SEM, where S. pyogenes
ATCC 19,615 cells appeared less aggregated, showed morphological changes, and reduced biofilm
formation. These observations are in accordance with the potential of phenolic compounds to damage
the membrane structure as well as anti-S. pyogenes properties of phytochemicals such as anti-adhesion,
anti-biofilm, and quorum-sensing inhibition [2,8]. The generation of ROS during the bio-reduction
of naphthoquinones [15] might be linked to the loss of cell wall integrity observed. In addition, the
visible effect of 1% DMSO on the bacteria, which do not appear on the growth (Figure 1), could also
slightly enhance the antibacterial effect of the phytochemicals.
Considering the potential clinical application of our study, additional experiments could be
conducted on combination of natural antibacterial agents and currently used antibiotics to enhance
the present management practices against S. pyogenes. If the combination demonstrates a synergic
and/or a complementary effect (e.g., anti-inflammatory and antitussive), it could become a combined
treatment for patient’s pain relief. Moreover, it is also possible to incorporate the efficacious natural
compounds in dehydrated honey lozenges. Honey itself has a recognized effect as being systemic
antitussive and antimicrobial natural source [18–20]. Interestingly, a recent study reported that honey
with a high phenolic content presented the highest antimicrobial activity [20]. Further investigations
are required to affirm the synergic effect with honey but also understand the safety of the use of
naphthoquinones in throat lozenges before making any recommendations for their use for managing
streptococcal pharyngitis.

Acknowledgments: This project was cofounded by the Natural Sciences and Engineering Council (NSERC) of
Canada and Island Abbey Foods Ltd, Charlottetown, PEI, Canada. We are grateful to R. Davidson (Queen Elizabeth
II Hospital, Halifax, NS, Canada) for providing the clinical isolate of S. pyogenes.
Author Contributions: Sabrina Macé and H.P. Vasantha Rupasinghe designed the study in collaboration with
Lisbeth Truelstrup Hansen. Sabrina Macé performed the experiments, analyzed the data, and wrote the manuscript.
H.P. Vasantha Rupasinghe and Lisbeth Truelstrup Hansen supervised the study and the manuscript writing.
Conflicts of Interest: The authors declare no conflict of interest.
Medicines 2017, 4, 25 8 of 9

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